Brown algae contain a variety of sulfated fucose-containing polysaccharides. For example, sulfated fucose-containing polysaccharides such as (1) sulfated fucans consisting of fucose and sulfate groups; (2) sulfated fucoglucuronomannans containing glucuronic acid, mannose, fucose and sulfate groups, e.g., the sulfated fucose-containing polysaccharide-U as described in WO 97/26896 (approximate molar ratio of constituting saccharides, fucose:mannose:galactose:uronic acid:sulfate group=10:7:4:5:20; hereinafter referred to as U-fucoidan); and (3) sulfated fucogalactan consisting of fucose and galactose, e.g., the sulfated fucose-containing polysaccharide-F as described in WO 97/26896 (approximate molar ratio of constituting saccharides, fucose:galactose=10:1; hereinafter referred to as F-fucoidan), or the sulfated fucose-containing polysaccharide-G as described in WO 00/50464 (approximate molar ratio of constituting saccharides, galactose:fucose=2:1; hereinafter referred to as G-fucoidan) are known. Almost all of these sulfated fucose-containing polysaccharides are macromolecular anions. Therefore, they behave in a chemically and physically similar manner in various purification steps, making it difficult to separate them from each other. For this reason, biological activities of sulfated fucose-containing polysaccharides derived from brown algae have often been examined without separating them from each other. Therefore, it was difficult to identify the sulfated fucose-containing polysaccharide that was responsible for the observed biological activity.
To date, correlation between an activity and a molecule is known for the sulfated fucan fraction as described in Agricultural and Biological Chemistry, 44 (8): 1965-1966 (1980), which is responsible for anticoagulant activity, and U-fucoidan as described in WO 97/26896, which is responsible for an apoptosis-inducing activity against tumor cells.
The use of the sulfated fucan fraction as an anticoagulant in place of heparin has been examined. However, it is required to obtain a highly pure sulfated fucan in order to use it as a pharmaceutical avoiding side effects due to unexpected activities. Thus, a method therefor has been desired.
Regarding U-fucoidan, it is similarly required to conveniently obtain a highly pure sulfated fucose-containing polysaccharide-U in order to prepare a pharmaceutical utilizing the apoptosis-inducing activity against tumor cells. Thus, a method therefor has been desired.
Generally, enzymatic degradation is the most efficient manner utilized for structural analyses of polysaccharides and production of oligosaccharides. Furthermore, only one polysaccharide can be readily removed from a mixture of polysaccharides which are hardly separated from each other as follows. The polysaccharide to be removed is converted into a smaller molecule using an enzyme that specifically degrades the polysaccharide. The mixture is then subjected to molecular weight fractionation such as ultrafiltration.
It has been reported that abalones, scallops, sea urchins, marine microorganisms and the like produce enzymes that degrade sulfated fucose-containing polysaccharides. However, only a trace amount of such an enzyme is generally contained in an organism. In addition, since such an organism has plural sulfated fucose-containing polysaccharide-degrading enzymes, various purification steps are required for obtaining a single enzyme. For example, the sulfated fucogalactan-degrading enzyme as described in WO 00/50464 was separated using its activity as an index, although it is unknown if it was isolated as a single protein. As described above, it is difficult to purify and collect a large amount of a single naturally occurring sulfated fucogalactan-degrading enzyme. Furthermore, it is necessary to add a sulfated fucogalactan or sulfated fucose-containing polysaccharides including a sulfated fucogalactan to a culture in order to obtain a sulfated fucogalactan-degrading enzyme from a marine microorganism. Thus, there are problems that the cultivation procedure is made complicated and the cost is made high.
There are further problems as follows. If a sufficient amount of a naturally occurring sulfated fucogalactan-degrading enzyme protein cannot be obtained as described above, it is almost impossible to obtain information about the amino acid sequence or the nucleotide sequence for the enzyme. Information about the N-terminal amino acid sequence might not be obtained due to blocking of the enzyme protein at the N-terminus even if a sufficient amount could be obtained.
In addition, there may be unexpected problems as follows. Even if a gene encoding such an enzyme could be obtained, the gene might not be expressed or the expression level might be low due to the incompatibility between the gene and the expression promoter or the host. Alternatively, a recombinant enzyme retaining an enzymatic activity might not be obtained due to formation of inclusion bodies.